The present invention relates to recombinant DNA which encodes the NheI restriction endonuclease as well as NheI methyltransferase, expression of NheI restriction endonuclease from E. coli cells containing the recombinant DNA.
NheI restriction endonuclease is found in the strain of Nisseria mucosa heidelbergensis (ATCC 25999). It cleaves double-stranded DNA G/CTAGC to generate a 4-base 5xe2x80x2 overhanging ends.
Type II restriction endonucleases are a class of enzymes that occur naturally in bacteria and in some viruses. When they are purified away from other bacterial proteins, restriction endonucleases can be used in the laboratory to cleave DNA molecules into small fragments for molecular cloning and gene characterization.
Restriction endonucleases act by recognizing and binding to particular sequences of nucleotides (the xe2x80x98recognition sequencexe2x80x99) along the DNA molecule. Once bound, they cleave the molecule within, to one side of, or to both sides of the recognition sequence. Different restriction endonucleases have affinity for different recognition sequences. Over two hundred and eleven restriction endonucleases with unique specificities have been identified among the many hundreds of bacterial species that have been examined to date (Roberts and Macelis, Nucl. Acids Res. 27:312-313, (1999)).
Restriction endonucleases typically are named according to the bacteria from which they are derived. Thus, the species Deinococcus radiophilus for example, produces three different restriction endonucleases, named DraI, DraII and DraIII. These enzymes recognize and cleave the sequences 5xe2x80x2TTT/AAA3xe2x80x2, 5xe2x80x2PuG/GNCCPy3xe2x80x2 and 5xe2x80x2CACNNN/GTG3xe2x80x2 respectively. Escherichia coli RY13, on the other hand, produces only one enzyme, EcoRI, which recognizes the sequence 5xe2x80x2G/AATTC3xe2x80x2.
A second component of bacterial restriction-modification (R-M) systems are the methyltransferase (methylases). These enzymes are complementary to restriction endonucleases and they provide the means by which bacteria are able to protect their own DNA and distinguish it from foreign, infecting DNA. Modification methylases recognize and bind to the same recognition sequence as the corresponding restriction endonuclease, but instead of cleaving the DNA, they chemically modify one particular nucleotide within the sequence by the addition of a methyl group (C5 methyl cytosine, N4 methyl cytosine, or N6 methyl adenine). Following methylation, the recognition sequence is no longer cleaved by the cognate restriction endonuclease. The DNA of a bacterial cell is always fully modified by the activity of its modification methylase. It is therefore completely insensitive to the presence of the endogenous restriction endonuclease. It is only unmodified, and therefore identifiably foreign DNA, that is sensitive to restriction endonuclease recognition and cleavage.
With the advent of recombinant DNA technology, it is now possible to clone genes and overproduce the enzymes in large quantities. The key to isolating clones of restriction endonuclease genes is to develop a simple and reliable method to identify such clones within complex genomic DNA libraries, i.e. populations of clones derived by xe2x80x98shotgunxe2x80x99 procedures, when they occur at frequencies as low as 10xe2x88x923 to 10xe2x88x924. Preferably, the method should be selective, such that the unwanted majority of clones are destroyed while the desirable rare clones survive.
A large number of type II restriction-modification systems have been cloned. The first cloned systems used bacteriophage infection as a means of identifying or selecting restriction endonuclease clones (EcoRII: Kosykh et al., Mol. Gen. Genet. 178: 717-719, (1980); HhaII: Mann et al., Gene 3: 97-112, (1978); PstI: Walder et al., Proc. Nat. Acad. Sci. 78: 1503-1507, (1981)). Since the presence of restriction-modification systems in bacteria enable them to resist infection by bacteriophage, cells that carry cloned restriction-modification genes can, in principle, be selectively isolated as survivors from genomic DNA libraries that have been exposed to phages. This method has been found, however, to have only limited value. Specifically, it has been found that cloned restriction-modification genes do not always manifest sufficient phage resistance to confer selective survival.
Another cloning approach involves transferring systems initially characterized as plasmid-borne into E. coli cloning plasmids (EcoRV: Bougueleret et al., Nucl. Acids. Res. 12: 3659-3676, (1984); PaeR7: Gingeras and Brooks, Proc. Natl. Acad. Sci. USA 80: 402-406, (1983); Theriault and Roy, Gene 19: 355-359 (1982); PvuII: Blumenthal et al., J. Bacteriol. 164: 501-509, (1985); Tsp45I: Wayne et al. Gene 202:83-88, (1997)).
A third approach is to select for active expression of methylase genes (methylase selection) PCT/US98/18124, filed Sep. 01, 1998; PCT/US99/13295, filed Jun. 11, 1999; U.S. Pat. No. 5,200,333, issued Apr. 6, 1993 and BsuRI: Kiss et al., Nucl. Acids. Res. 13: 6403-6421, (1985)). Since R-M genes are often closely linked together, both genes can often be cloned simultaneously. This selection does not always yield a complete restriction system however, but instead yields only the methylase gene (BspRI: Szomolanyi et al., Gene 10: 219-225, (1980); BcnI: Janulaitis et al., Gene 20: 197-204 (1982); BsuRI: Kiss and Baldauf, Gene 21: 111-119, (1983); and MspI: Walder et al., J. Biol. Chem. 258: 1235-1241, (1983)).
A more recent method, the xe2x80x9cendo-blue methodxe2x80x9d, has been described for direct cloning of restriction endonuclease genes in E. coli based on the indicator strain of E. coli containing the dinD::lacZ fusion (Fomenkov et al., U.S. Pat. No. 5,498,535, (1996); Fomenkov et al., Nucl. Acids Res. 22:2399-2403, (1994)). This method utilizes the E. coli SOS response following DNA damages caused by restriction endonucleases or non-specific nucleases. A number of thermostable nuclease genes (TaqI, Tth111I, BsoBI, Tf nuclease) have been cloned by this method (U.S. Pat. No. 5,498,535, (1996).
Because purified restriction endonucleases, and to a lesser extent, modification methylases, are useful tools for creating recombinant molecules in the laboratory, there is a commercial incentive to obtain bacterial strains through recombinant DNA techniques that produce large quantities of restriction enzymes. Such overexpression strains should also simplify the task of enzyme purification.
Nisseria mucosa heidelbergensis genomic DNA was digested partially with ApoI and genomic DNA fragments in the range of 2-10 kb were purified through a low melting agarose gel. The ApoI partial DNA fragments were ligated to EcoRI digested and CIP treated pRRS vector skolund, Gene 88:1-5 (1990). The ligated DNA was transferred into E. coli RR1 competent cells by electroporation. Transformants were pooled and amplified. Plasmid DNA was prepared from the cells and challenged with BfaI. BfaI recognition sequence C/TAG is the internal 4 bp of NheI recognition sequence G/CTAGC. It was reasoned that cloning and expression of NheI methylase may confer resistance to BfaI digestion. The BfaI challenged DNA was transformed into RR1 cells. Survivors were screened for resistance to NheI digestion. However, no NheI resistant clones were detected.
In order to clone the NheI methylase gene (nheIM), one NheI linker was inserted into SspI and HincII sites respectively in pRRS. The modified plasmid with two NheI sites was named pRRS10. Vector pRRS10 was digested with EcoRI, treated with CIP, and gel-purified. The ApoI partial DNA fragments were ligated to pRRS10 with compatible ends. The methylase selection method was used to clone nheIM gene. Ten resistant clones were isolated from the ApoI partial library. One resistant clone #22 contains an insert of about 2.8-3 kb. The entire insert was sequenced by primer walking. The nheIM gene is 882 bp, encoding a 293-aa protein with predicted molecular mass of 33,268 daltons.
There is one gene downstream of nheIM gene that has 43% identity to a 20 kDa hypothetical protein (O47152) of E. coli . There is one partial open reading frame (orf) upstream of nheIM gene that doe not show any homology to known genes in GenBank. Since restriction endonucleases usually are not homologous to each other (except among some isoschizomers), it was concluded that the upstream orf is most likely the nheIR gene. Efforts were made to obtain the upstream DNA coding sequence by inverse PCR amplification. N. mucosa heidelbergensis genomic DNA was digested with AluI, HaeII, HhaI, NheI, NlaIII, NspI, SphI, SspI, StyI, TaqI, and TfiI, respectively. The digested DNA was self-ligated at a low DNA concentration and then used for inverse PCR amplification of the nheIR gene. Inverse PCR products were found in AluI, NlaIII, NspI, StyI, and TaqI digested/self-ligated DNA. The inverse PCR products were gel-purified and sequenced which provided 339-bp new DNA sequence. A second round of inverse PCR was performed to amplify more of the upstream sequence. Inverse PCR products were found in ApoI, AseI, RsaI, SspI, and TaqI digested/self-ligated DNA. The PCR products were sequenced directly using PCR primers. An additional 329-bp new sequence was derived. One open reading frame of 987 bp was found upstream of nheIM gene. This orf was designated as nheIR gene, which encodes a 328-aa protein with predicted molecular mass of 38,197 daltons.
Two primers were synthesized to amplify nheIM gene in PCR. Following digestion with BamHI and SphI, the PCR product was ligated into pACYC184 with compatible ends. Plasmids with nheIM gene inserts were screened for resistance to NheI digestion. Eleven clones out of 18 were resistant to NheI digestion, indicating efficient M.NheI expression in E. coli cells and NheI site modification on the expression plasmid. The host cell ER2683 [pACYC-NheIM] was used for expression of nheIR gene.
The nheIR gene was amplified in PCR, digested with BamHI and ligated into pUC19lacIq or pAII17. The expression level from pUC19lacIq-NheIR was approximately 10,000 units of NheI per gram of wet cells. The native NheI producing strain N. mucosa heidelbergensis produces about 100,000 units of NheI per gram of wet cells. So the NheI yield in the first recombinant strain is about 10-fold lower than the native strain. The second expression strain carrying pAII17-NheIR1 produces approximately 100,000 units of NheI, which is about the same level as that produced in the native strain N. mucosa heidelbergensis. To further improve the NheI expression level, the internal NdeI site within nheIR gene was mutagenized by introduction of a silent mutation. A new NdeI site was engineered at the beginning of the gene in the forward primer. A BamHI site was introduced in the reverse primer. The nheIR gene was amplified in PCR, digested with NdeI and BamHI and cloned into the T7 expression vector pAII17. The fusion of ATG start codon of nheIR gene to the NdeI site in the expression vector increased NheI expression level to 10 million units NheI per gram of wet E. coli cells. The recombinant NheI expression level in the E. coli overproducing strain is 100-fold higher than the native source.